Data Mining Of Genes Related To Endometrial Receptivity And The Study Of S100P In The Establishment Of Endometrial Receptivity

Embryo implantation, as the most relevant limiting factor for successful pregnancy, is a critical step in pregnancy. The formation of endometrial receptivity is requisite for successful implantation. Physiologically, the human endometrium is receptive on the19-24th day of a menstrual cycle. This transitory period is called "implantation window", during which the endometrium undergoes extensive morphological and physiological changes to facilitate implantation of the embryo under the accurate regulation of the hypothalamic-pituitary-ovarian (H-P-O) axis. As one of the indispensable factor, the endometrial receptivity composes the major factor limiting assisted reproductive technologies (ART) and unexplained infertility. Therefore, the diagnosis and evaluation of endometrial receptivity has very important clinical significance, whitch will greatly promote the progress of the reproductive medicine.The past two decades have witnessed an explosive growth in the amount of genomic studies, which resulted in unprecedented amount of data accumulated. The same was true in the studies of endometrial receptivity. However, due to the nature of the data, variability, small sample volume and high dimension size, the results of high-throughput data were unreliable. Up to now, the reliable diagnostic markers are still lacking, the molecular mechanism under the endometrial receptivity remains unclear and the mass data are far from being fully used. The emergence of bioinformatics gives us the possibility to deal with this problem. It can not only avoid redundancy and unnecessary waste but also acquire more reliable results. We employed bioinformatics tools integrating microarray data related to endometrium receptivity from multi-center, mining potential biomarkers and further deciphering the functions and interactions of these molecules. Our studies provided a lot of valuable clues for further studies.Embryo implantation is a complex process that involves embryo apposition and attachment to the maternal endometrial epithelium, traversing adjacent cells of the epithelial lining and invasion into the endometrial stroma. There are many biological processes similar to tumors, such as cell proliferation, apoptosis, adhesion, invasion and angiogenesis. S100P, a small molecular calcium binding protein highly expressed in various tumor cells, participated in the proliferation, survival, metastasis and invasion of tumor cells, and related to the development, prognosis, and drug resistance of tumors, has been selected out specially highly up-regulated in the window of implantation in human endometrium in our previous works. It prompts that S100P may play a role in embryo implantation, however few publications focus on the role and mechanism of S100P in embryo implantation. We applied knock-down and overexpression to study the role and mechanism of S100P in the formation of endometrial receptivity, whitch will help us better understanding the mechanisms of human embryo implantation.Part One Data mining of genes related to endometrial receptivityPurpose:1. To mine potential biomarkers of the receptive endometrium by bioinformatics tools.2. Analyzing the functions and interactive networks of the potential biomarkers to decipher the mechanism of endometrial receptivity.3. Using clinical samples to substantiate the results of data mining.Materials and methods:1. CEL files were collected from public microarray data repositories and then uploaded onto GeneSifter to mining potential biomarkers whichspatial-temporally expressed in the mid-secretory phase of endometrium.2. The differentially expressed genes were then uploaded onto IPA for function and interactive network analysis.3. Endometrial samples from normal fertile women were collected and then realtime PCR was employed to verify the expression pattern of selected genes through the menstrual cycle.Results:1.1543gene sets were found to express differentially, of which148highly regulated genes were listed as potential biomarkers of the receptive endometrium.2. The function and pathway analysis identified the differentially expressed genes primarily involved in the immune response and cell cycle. Two networks related to the cardiovascular system and cancers were generated within the genes which changed more than10-fold. 3. Nine genes (DKK1, S100P, FAM148A, MAOA, SFRP4, CXCL14, CRISP3, ANXA4and SFN) were validated by real-time PCR.Conclution:It was a meaningful exploration of the existing data to acquire useful and reliable information, and our results undoubtedly provided valuable clues for further studies. Part Two The role and mechanism of S100P in the formation of endometrial receptivityPurpose:1. To examine the expression pattern of S100P in endometrium through the menstrual cycle and in placenta through the pregnancy.2. To investigate the hormonal regulation of S100P.3. To study the biological function of S100P in the establishment of endometrial receptivity and embryo implantation.4. To probe the potential molecular mechanism of S100P in the establishment of endometrial receptivity and embryo implantation.Materials and methods:1. Endometrium samples of different menstrual cycle were collected from normal fertile women and placental tissues were collected from nomal pregnant women. Realtime PCR, western blot and immunofluorescence were used to evaluate the expression and location of S100P.2. In vitro cultured endometrial cells, including primary endometrial stromal cells, epithelial cells and cell lines RL952and Ishikawa, were treated with various concentrations of17-beta-estradiol (E2), progenterone (P4) and HCG for different periods. Realtime PCR and in-cell western were employed to investigate the expression change of S100P.3. The expression of S100P was overexpressed in primary stromal cells and inhibited in endometrial cell line RL952and Ishikawa by lentivirus system. Cell migaration was detected by scratch test and cell invasionwas dctected by transwell assays.4. RL952or primary endometrial stromal cells-Bewo cell sphere co-culture model were established to investigate the effects of S100P to the adhesion of Bewo cell sphere in RL952cell monolayers or theinvasion of Bewo cell sphere in primary endometrial stromal cell monolayers.5. Phalloidin staining was used to observe the reorganization of cytoskeleton, Realtime PCR and immunofluorescence were employed to study the activation of|3-catenin and NFkB signaling pathway after overexpressing or inhibiting S100P in endometrial cells.Results:1. S100P was spatial-temporally up-regulated in the endometrium of mid-secretory phase during the menstrual cycle. And in the process of pregnancy, S100P was highly expressed in placenta of late pregnancy and early gestation villi?2. The expression of S100P could be up-regulated by estrogen, progesterone and HCG in primary stromal and epithelial cells and Ishikawa cell lines. And the regulation S100P in primary stromal cells was displayed in a time-and concentration-depend way.3. Expression of S100P mRNA and protein in RL952and Ishikawa cells were strongly inhibited by over98%. Expression of S100P mRNA and protein in primary stromal cells were overexpressed more than5-fold. S100P inhibited the migaration of endometrial cells but promoted their invasion. The overexpression of S100P facilitated the invasion of Bewo sphere in ESCs, and the RNA interference of S100P inhibited the adhesion of Bewo sphere to RL952monoplayer.4. The interference of S100P in RL952promoted the F-actin gathered in cell membrane. And the overexpression of S100P in ESCs inhibited the translocation of β-catenin into nucleus, but promoted the translocation of NFkB into nucleus and up-regulated the mRNA expression of IKKB.Conclusions:1. S100P was highly up-regulated in the endometrium in both epithelium and stoma during the window of implantation.2. The expression of S100P in endometrial cells could be regulated by E2, P4and HCG.3. S100P promoted the invasion but inhibited the migaration of endometrial cells.4. S100P increased the adhesion rate of embryo to the monoplayer of RL952and facilitated the expansion of Bewo spheres in the monoplayer of ESCs.5. S100P might influence β-catenin and NFkB signaling pathway involved in the establishment of endometrial receptivity.